I propose to exploit the absorbance spectrum of the pH-sensitive dye carboxyfluorescien (CF) to measure intracellular pH (pH(i)) in the isolated, perfused proximal tubule of the rabbit. My goal is to individually study luminal and basolateral (i.e., "blood-side") transport of H+ and HCO(3)-, first in the cells of the proximal convoluted tubule (PCT) and then in the proximal straight tubule (PST). The first step will be to validate this optical technique on the isolated, perfused proximal tubule of the salamander, a preparation in which + and HCO3- transport has already been studied with pH-sensitive microelectrodes. I will use such microelectrodes to obtain an intracellular calibration of CF both in the steady state, and during rapid changes in pH(i). I will then begin a detailed analysis of presumably luminal) H+ and (presumably basolateral) HCO3 transport in the rabbit PCT. In the case of H+ transport, the approach will be to apply an intracellular acid load using the NH4+ technique, while monitoring the subsequent recovery of pH(i). (Basolateral HCO3 transport will be minimized by reduction of HCO3-). The PH(i) recovery rate is directly related to the rate of H+ extrusion (presumably at the luminal membrane), which if due to Na-H exchange, should be reduced by lowering luminal (Na+) or adding amiloride. In the case of HCO3- transport, I will hold pCO2 constant while simultaneously lowering basolateral (HCO3) and pH. This should produce a fall in pH(i) due to basolateral HCO3- efflux; the efflux should be blocked by stilbene derivatives and possibly by carbonic anhvdrase inhibitors. These studies will be extended to the PST. Technically, the CF will be introduced into the cells as a precursor which is metabolized to CF. The intracellular absorbance spectrum of CF will be followed in real time by a spectrophotometer capable of obtaining an entire spectrum in 16 ms. Thus, I will be able to monitor the very rapid pH(i) changes expected in these mammalian cells. My long-term goal is to use this optical approach to characterize H+ and HCO3- transport, and the regulation thereof, along the entire mammalian mephorn, relating this new data to known properties of renal acid secretion.